Activating c-kit gene mutations in human germ cell tumors.

Abstract

The c-kit gene encodes a tyrosine kinase receptor (KIT) that is required in normal spermatogenesis and is expressed in seminomas and dysgerminomas, a subset of human germ cell tumors (GCTs). To determine whether activating mutations of the c-kit gene occur in GCTs, primary tissue samples of 33 testicular and ovarian tumors were examined for mutations in the juxtamembrane and phosphotransferase domains by polymerase chain reaction amplification and DNA sequencing. A novel missense mutation (D816H) was found in the phosphotransferase domain in tumors of seminoma/dysgerminoma differentiation. The c-kit alleles in nonneoplastic tissues from these patients were wild type, suggesting that the mutant alleles were acquired and selected for during malignant transformation. In cell transfection experiments, the D816H mutant protein was a constitutively activated kinase and was constitutively phosphorylated on tyrosine residues. This is the first description of an activating c-kit mutation in GCTs and is evidence that the KIT signal transduction pathway is important in the pathogenesis of neoplasms with seminoma differentiation.

Autoradiographs of DNA sequencing. Cycle sequencing of c-kit PT domain PCR amplified products from GCTs (designated as T) and from nonneoplastic tissue (designated as N) are shown. In nonneoplastic tissue only a wild-type sequence is present; in tumor samples mutant and wild-type sequences are coexistent, consistent with retention of one wild-type allele in the tumor cells. In all tumor samples there is a G-to-C substitution at position 2467 (solid arrows), resulting in an amino acid coding substitution (D816H). A: The two components of a mixed ovarian GCT, a yolk sac tumor component (designated as T1) and a dysgerminoma component (designated as T2), were sampled separately. Both tumor components contain the G-to-C substitution resulting in the D816H mutation (solid arrow). In addition, there is a C-to-T substitution at nucleotide position 2469 (open arrow), which does not result in a change in amino acid coding. B: Results from a seminoma showing the G-to-C substitution at position 2467 that results in the D816H amino acid missense mutation.

The D816H KIT gene product is a constitutively activated kinase. COS cells were transfected with eukaryotic expression vector constructs containing wild-type and mutant forms of c-kit, as well as the vector alone as a negative control. A:In vitro kinase assay. Protein extracts of transfectants were immunoprecipitated with anti-KIT antibodies and incubated with [γ-32P]ATP. The upper panel shows an autoradiograph of a blot of the kinase assays of equal amounts of protein extracts after separation by gel electrophoresis. The location of molecular mass standards are shown to the left in kd. The location of major phosphorylation targets are consistent with KIT protein. Both the well-characterized D816V mutant and the D816H mutant show a marked increase in kinase activity when compared to wild-type KIT. The same blot was probed with anti-KIT antibody to control for levels of KIT protein in the extracts (lower panel). Similar levels of D816V and D816H mutant KIT proteins were expressed by the transfected cells, with higher levels of expression of wild-type KIT. B: Anti-phosphotyrosine immunoblot. Protein extracts of transfectants were immunoprecipitated with anti-KIT antibodies, separated by gel electrophoresis, and blotted. The upper panel shows a blot probed with anti-phosphotyrosine antibody. A protein is detected at the appropriate molecular mass for KIT in extracts containing D816V and D816H KIT, but not wild-type KIT. The same blot was stripped and reprobed with anti-KIT antibody to control for the presence and quantity of KIT proteins (lower panel).